Chromosomal abnormalities detected by karyotyping among patients with secondary amenorrhea: a retrospective study

ABSTRACT BACKGROUND: Chromosomal abnormalities (CAs) have been described in patients with secondary amenorrhea (SA). However, studies on this association are scarce. OBJECTIVES: To evaluate the frequency and types of CAs detected by karyotyping in patients with SA. DESIGN AND SETTING: This retrospective study was performed in a reference clinical genetic service in South Brazil. METHODS: Data were obtained from the medical records of patients with SA who were evaluated between 1975 and 2022. Fisher’s bicaudate exact test and Student’s t-test were used, and P < 0.05 was considered significant. RESULTS: Among 43 patients with SA, 14 (32.6%) had CAs, namely del (Xq) (n = 3), 45,X (n = 2), 46,X,r(X)/45,X (n = 2), 46,XX/45,X (n = 1), 46,X,i(q10)/45,X (n = 1), 47,XXX (n = 1), 46,XX/47,XXX (n = 1), 46,XX/47,XX,+mar (n = 1), 45,XX,trob(13;14)(q10;q10)/46,XXX,trob(13;14)(q10;q10) (n = 1), and 46,XX,t(2;21)(q23;q11.2) (n = 1). Additional findings were observed mostly among patients with CA compared with those without CA (P = 0.0021). No difference in the mean age was observed between the patients with SA with or without CAs (P = 0.268025). CONCLUSIONS: CAs are common among patients with SA, especially those with short stature and additional findings. They are predominantly structural, involve the X chromosome in a mosaic, and are compatible with the Turner syndrome. Patients with SA, even if isolated, may have CAs, particularly del (Xq) and triple X.


INTRODUCTION
Amenorrhea is a symptom, not a proper condition, characterized by an alteration in the menstrual cycle that affects 2%-5% of women of childbearing age. 1,2 Secondary amenorrhea (SA) corresponds to most amenorrhea cases and affects 3%-4% of women of childbearing age. It is defined by the cessation of menstruation for a minimum period of 3 months in patients with previously regular cycles or 6 consecutive months in women who have had at least one previous menstruation. 1,3 The diagnostic evaluation of patients with SA begins with assessing patient history, followed by conducting physical examination, laboratory tests, and imaging. 4,5 Although karyotyping is not routinely performed, it can be an important test.
Considering hormones, SA can be classified as having a central (hypothalamic-hypophyseal) or peripheric (ovarian) origin. 1,6 Hypothalamic disorders are some of the most common causes of amenorrhea, including SA. 7 SA might also have a peripheric origin because of a primary ovarian insufficiency that occurs after menarche and before the age of 40 years. 8 Several factors can be related to SA, including genetics, the environment, and the interactions between them. Among genetic causes, chromosome abnormalities (CAs), 1,6 which are generally identified by cytogenetic tests (e.g. karyotyping) are observed. Most CAs are chromosome X-related, similar to the Turner syndrome (TS). 6,9 Thus, determining the cause of SA is essential for the appropriate management and treatment of patients. 5 However, studies on SA and CAs are few, and most of them are case reports or case series. 9 The patients were also classified as either syndromic or non-nonsyndromic by a single clinical geneticist. Additionally, the patients were divided into those with hypogonadotropic hypogonadism or hypergonadotropic hypogonadism. 6  were diagnosed with TS, and 3 (7%) had triple X syndrome.
When comparing the groups with and without CAs, those with CAs only had more additional findings (P = 0.0021). We did not identify significant differences in mean age at the first evaluation (P = 0.9612), mean age of SA (P = 0.2680), periods between age at menarche and cessation of menstruation (P = 0.4285), hormonal profile (P = 1.0000), or syndromic aspect (P = 0.5855) ( almost all the patients in our sample were referred from). Therefore, they were selected before the clinical genetics evaluation.
CAs described in association with SA can be numeric or structural, and they can occur in an isolated form or involve more than one cellular lineage. 6,9 Furthermore, these patients generally exhibit a wide range of phenotypic abnormalities, other clinical features, and hormonal profiles. 15 In the literature, CAs associated with SA have been described to mainly affect the X chromosome, 9 a finding that accords with that of our study reporting that among patients with CAs, 85.7% had abnormalities involving the X chromosome. In our sample, the main alteration involving the X chromosome was the deletion of parts of its long arm, which corresponded to 21.4% of the CA cases.
Proper functioning of the gonads depends on the integrity of both X chromosomes. 9 A region of great importance related to normal ovary development and functioning is localized at the long arm of the X chromosome and ranging from Xq13.3 to Xq27. 16  were diagnosed with ovarian failure at 27 and 28 years old, respectively. Meanwhile, the patient with the deletion involving the POF2 (region q13q26) was diagnosed with ovarian failure at 15 years old.
As previously mentioned, these findings are consistent with those in the literature. 11,18 The genes placed in the POF1 and POF2 loci of    [19][20][21][22][23][24][25][26] However, despite the description of all these candidate genes, the cause of premature ovarian failure remains unknown in most cases. 27,28 In our study, 14% of the patients had TS, a condition characterized by total or partial absence of the X chromosome. As observed in our sample, it can present as different chromosomal constitutions. 17 It occurs in approximately 1 in 2,500-5,000 women and is commonly diagnosed later, on average at the age of 15 years. 29 Patients with this syndrome may have typical clinical characteristics that include cardiac, skeletal, and endocrine abnormalities, including hypothyroidism and short stature. 17,30 However, this clinical spectrum ranges from a typical appearance to a presentation without clinical characteristics or minimal findings. 30 These characteristics and clinical variability were also observed in our sample, in which the most consistent finding was short stature, which was present in all the patients. Moreover, of the patients with short stature in our sample, 71.4% were diagnosed with TS, and this finding was associated with the presence of the syndrome. Thus, a short stature in TS is due to the haploinsufficiency of the SHOX gene, which is located on the short arm of the X chromosome. 31 Post-pubertal patients with TS commonly present with hypergonadotropic hypogonadism due to ovarian dysgenesis that leads to premature ovarian failure. Therefore, most patients experience pubertal delay and primary amenorrhea. However, SA has also been observed, especially when associated with mosaicism. 17 TS may also be caused by short-arm monosomy of the X chromosome. 17 In our sample, 3 of 6 patients with TS had this chromosome particularity. Two of them had the ring form of the X chromosome [r(X)] and mosaicism (with an associated 45,X lineage). The ring form of the X chromosome occurs because of the deletion of parts of its short and long arms, along with their posterior fusion. This constitution is related to atypical and severe cases of TS, including intellectual deficiency. This may occur because of XIST changes, which are the main genes responsible for controlling X chromosome inactivation. Therefore, modifications involving this region cause a greater expression of chromosomal material, leading to a higher frequency of abnormalities, including atypical abnormalities, such as microcephaly, agenesis of the corpus callosum, and seizures. The size of the ring X chromosome lacking XIST and, therefore, unable to become inactivated, correlates with phenotype severity in some cases. By contrast, patients with large rings that undergo selective X-chromosome inactivation are frequently associated with a more normal phenotype. 36 Most patients with ring X chromosomes are infertile, as are other patients with TS. The gonads comprise striae with no follicular development. However, some patients are fertile and may transmit the ring X-chromosome to their progeny. In these rare cases, the ring is commonly large, with breakpoints on the short arm at bands p13 and p22. On the long arm, the breakpoints are at band q24 or q27. 36 In our study, patients with ring X chromosome did not present atypical clinical features (possibly due to mosaicism with the associated 45,X lineage). One patient presented with hypothyroidism; however, as mentioned previously, this is a common finding in TS. The age of menstrual cycle cessation was low, as observed in patients with a 45,X constitution, and ranged from 15 to 16 years. This premature age of SA might be influenced by the 45,X lineage present in association with the ring X chromosome lineage that was observed in both patients.
Approximately 20%-30% of patients with TS are carriers of an isochromosome of the X chromosome long arm. 17 This finding was observed in one patient in our sample in association with a 45,X lineage. Patients with isochromosomes commonly present with clinical features similar to those of patients with a 45,X constitution. However, they have a higher frequency of dysgenetic gonads, primary amenorrhea, SA, short stature, and major anomalies, such as congenital heart defects and renal malformations. Autoimmune diseases, such as Hashimoto thyroiditis, are also common in these cases. 37 The patient with an X long arm isochromosome in our sample had a short stature without any associated malformations or autoimmune diseases.
Her menstrual cycle was interrupted at 24 years of age, which is an age older than that commonly described among patients with TS.
As previously mentioned, TS may occur in a mosaicistic constitution. The most common constitution is associated with a normal cellular lineage, 17 as detected in one patient in our sample.
This constitution is observed in 15% to 25% of TS cases. 17 These patients commonly present with a higher stature, a lower frequency of major abnormalities, and, most commonly, SA, when compared to those with a 45,X karyotype. Menarche has been described in approximately 2%-5% of cases. These findings may be attributable to the presence of a normal cell lineage. 9 Triple X syndrome is characterized by an extra X chromosome resulting from the nondisjunction of sexual chromosomes during the first meiotic division, and it occurs in 1 in every 1,000 women.
This alteration is highly related to advanced maternal age. The phenotype observed in each individual with triple X might vary, and only approximately 10% of the cases are diagnosed. 38 Although sexual development and ovarian function are normal in most of these patients, ovarian dysfunction may manifest as premature menopause, SA, or oligomenorrhea. 13,39 The first report of triple X syndrome involving a 35-year-old woman with SA was by Jacobs et al. 40 In our study, one patient had triple X syndrome and hypergonadotropic hypogonadism due to premature ovarian failure. SA was her only finding, and her menstrual cycle was interrupted at 19 years of age.
Although more rarely (10% of cases), triple X might also occur in mosaicism. 41 Temoçin et al. 42  in both lineages cannot be disregarded. Interestingly, Bertini et al. 44 have described a patient with similar clinical findings and uniparental maternal disomy of chromosome 14, which was also associated with Robertsonian translocation between chromosomes 13 and 14.
Chromosome markers comprise a few structurally abnormal chromosomes of unknown origin. One patient with mosaicism involving one normal cellular lineage with a supernumerary marker chromosome (47,XX+mar) was observed in our sample. In these cases, the use of molecular cytogenetic techniques, such as fluorescent in situ hybridization, is recommended to determine the marker chromosome origin. 17 However, our patient did not undergo this test since she was evaluated 20 years ago. As for her clinical condition, she did not have a syndromic appearance, and SA was the only finding.
Interestingly, one patient in our sample had an apparently balanced de novo translocation involving chromosomes 2 and 21 with normal parental karyotypes. The patient had an intellectual deficit associated with SA. First, no genes associated with her clinical condition were located at translocation breakpoints in the literature. Additionally, at the time of evaluation, molecular cytogenetic techniques were still unavailable.
The frequency of mosaicism identified in our study (50%) and that described in the literature (40%-100%) are both high. 6,42,43,45 It should also be recalled that most patients with TS presenting with SA have a mosaic constitution. 17,30 Hence, among the cases of SA, the number of analyzed cells in karyotypic evaluation should be higher, targeting the detection of potential mosaicism (this is in agreement with the higher cell count that is routinely performed in mosaicism suspicion, that is, in general, 100). 46

CONCLUSION
As CAs are common among patients with SA, cytogenetic analysis by karyotyping is important, especially in patients with short stature and additional findings. The main types of CAs observed were structural, involving the X chromosome, and compatible with a TS diagnosis. Several CAs occurred in the mosaic. This finding is also commonly reported in previous studies, which have suggested that patients with SA should be evaluated by karyotyping with more cells counted as a precaution.
However, the absence of additional findings other than SA did not exclude karyotype indication. This is because a significant number of patients, even those with CAs, may have SA as an isolated finding, especially in patients with the Xq deletion and triple X syndrome (with or without mosaicism). In our sample, we did not observe a difference in SA age between patients with and without CAs. However, we cannot rule out the influence of the small sample size on this result.
Therefore, the diagnosis of CAs, especially when performed in the early stages, is possible and important for better management of patients with SA.